Heparin Controlled Stimulation of in vivo
Angiogenesis Through Dual Growth
Factor Loaded Hyaluronic Acid Hydrogels
Peter Fuegy
Dr. Robert Peattie
Celeste Riley, Dan Pike, Kyle Pomraning
Dept. of Chemical Engineering
Background

Tissue Engineering
http://www.chemeng.unimelb.edu.au/images/sideimages/r_chem_eng5.jpg

Surgical Implants
http://www.vitekres.com/images/IMG0048.JPG

Why Are They Unsuccessful?
Terms to know

HA – Hyaluronic Acid
http://europa.eu.int/comm/research/press/images/fig01.jpg

PEGDA – polyethylene glycol diacrylate
Terms to know

VEGF – Vascular Endothelial
Growth Factor
http://www.find-a-drug.org.uk/images/1flt-q4.jpg

Ang-1 – Angiopoietin-1

Hydrogels
http://medweb.bham.ac.uk/vascularbiology/pics/ang1_s.jpg
Heparin (Hp)

Role:
–
–
–

Glycosaminoglycan in the extracellular matrix
Regulates thrombosis & blood vessel formation
Anti-tumor agent
Why Hp?
–
–
Hp-binding growth factors
Chemically modified Hp-DTPH
http://www-cryst.bioc.cam.ac.uk/~dima/images/all-heparin.gif
Goal

The project goal is to determine
whether the co-delivery of the growth
factors VEGF and Ang-1 in HA/Hphydrogel films can induce a
controlled angiogenic response in
vivo
Angiogenesis

Angiogenesis is the process by which new capillary
vessels grow and develop from preexisting vessels
http://www.med.unibs.it/~airc/angio2.gif

It can be stimulated via more than 24 GF’s as well
as low molecular weight HA
Previous Research

The Peattie Laboratory has shown that:
–
–
–
HA films are capable of storing and releasing GF’s
over a prolonged period of time
HA films produce a strong angiogenic response in
vivo
With Hp present HA films have been shown to
regulate the release of GF’s in vitro.
Summer Outline
2)
1)
3)
Data Analysis
Count Slides
Neovascularization Index
Experimental Control Setup
7 Days
14 Days
Sham 1 2 3 4 Sham 1 2 3 4
HA/Hp
-
+ + + +
-
+ + + +
VEGF
-
- + - +
-
- + - +
Ang-1
-
- - + +
-
- - + +
Making HA-Hydrogels

Step 1:
–
–
Chemically modified HA and Hp are dissolved into PBS pH 7.4
GF’s are dissolved in as needed
VEGF
Ang-1
VEGF Ang-1
Hp-DTPH
HA-DTPH
PBS
The solution is now
ready for crosslinking
Making HA-Hydrogels

Step 2:
–
Crosslink HA/HP and GF’s with PEGDA
Making HA-Hydrogels

Here is the process of
crosslinking

After a period of 24
hours the gels are fully
set and ready to
undergo dehydration
and form the film used
for implantation
Surgical Procedures

The HA/Hp-gel implants will be evaluated in vivo in male
Balb/C mice between 6 and 8 weeks of age
http://old.mendelu.cz/~agro/af/lme/image/Mice/Mouse-w.gif
Surgical Procedures



The mice are fully anesthetized and a 4-5mm
incision is made into the right ear pinna posterior
surface
A pocket is opened up and the implant is placed into
the pocket
After the trial time period both the contralateral and
tested ear are collected and tissue samples are
plated
Here are two samples of the plated tissues
(pictures were taken at 400X)
This slide
represents the
contralateral
ear
This slide
represents the
treated ear
Data Analysis

Count the vessels on each slide
–
An area of 25µm2 is used when counting
–
Each ear sample is counted 10 times, in
10 random locations to ensure the least
bias possible
Data Analysis
Contralateral, HA + VEGF
Treated, HA + VEGF
(14 days, H&E, x 400)
(14 days, H&E, x 400)
Data Analysis

Count the vessels on each slide
–
–

An area of 25µm2 is used when counting
Each ear sample is counted 10 times, in 10
random locations to ensure the least bias possible
Response or Random
–
How can this be determined?
Data Analysis

The neovascularization index (NI)
represents a quantifiable way to
account for any outside influences on
vessel growth
(treatment  CL)  ( sham  CL)
NI 
mean CL
–
–
–
treatment: Vessels counted on trial ear
CL: Vessels counted on contralateral ear
sham: Vessels counted with no implant
Results


Tissue samples are being plated
We expect results by late September
–
–
Data must be analyzed and compared to previous
experiments
NI values are compared
Acknowledgements

Howard Hughes Medical Institute

Dr. Robert Peattie

Celeste Riley

Dr. Kevin Ahern

Dr. Joseph McGuire

The University of Utah’s Prestwich Laboratory